Project Summary This application is for a predoctoral fellowship awarded by the National Cancer Institute to physician-scientist trainees. The applicant is a trainee of the Medical Scientist Training Program at the Perelman School of Medicine at the University of Pennsylvania. This award would help him achieve his career goal of becoming an academic physician-scientist Principal Investigator studying genome maintenance in cancer. Cancer cells must maintain their telomeres in order to achieve replicative immortality. Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism used by 10-15% of human cancers and often portends a poor prognosis. Although ALT occurs via a homologous recombination (HR)-based mechanism, the essential steps of DNA synthesis that culminate in telomere copying and elongation remain poorly understood. Studies in yeast suggest that ALT synthesis may occur by break-induced replication (BIR), a non-canonical and mutagenic form of DNA synthesis that is also implicated in complex genomic rearrangements seen in cancer. We have previously published a unique system capable of inducing ALT activity. Using this system, along with baseline ALT+ cells I will mechanistically dissect ALT synthesis. In this proposal I provide details of 2 aims that will guide thorough testing of my hypotheses. Briefly, in Aim 1, I will determine the role of POLD3, a polymerase subunit implicated in BIR, as well as other replication and HR factors in ALT synthesis through testing the effects of their depletion on bromodeoxyuridine (BrdU) incorporation at telomeres. Additionally, I will interrogate the factors governing the recruitment of POLD3 to ALT telomeres. In Aim 2, I will define the mode of DNA synthesis during ALT using next-generation sequencing of nascent DNA and telomeric DNA combing to ascertain replication parameters including mutagenicity and processivity. In summary, I intend to use ALT as a system to answer fundamental questions about DNA synthesis during repair, including polymerase involvement and basic characteristics. The expected results have broad implications for genome maintenance and cancer mechanisms, and will facilitate the development of novel therapeutics targeting ALT and non- canonical synthesis.